Initially, the successful culture of mammalian cells in vitro required supplementation of growth medium with serum which provides hormones and growth factors necessary for cell attachment and proliferation. Although serum is still widely used for mammalian cell culture, there are several problems associated with its use (Freshney, Serum-free media. In Culture of Animal Cells, John Wiley & Sons, New York, 91-99, 1994): 1) serum contains many unidentified or non-quantified components and therefore is not "defined"; 2) the composition of serum varies from lot to lot, making standardization difficult for experimentation or other uses of cell culture; 3) because many of these components affect cell attachment, proliferation, and differentiation, controlling these parameters, or studying the specific requirements of cells with respect to these parameters, is precluded by the use of serum; 4) some components of serum are inhibitory to the proliferation of specific cell types and to some degree may counteract its proliferative effect, resulting in sub-optimal growth; and 5) serum may contain viruses which may affect the outcome of experiments or provide a potential health hazard if the cultured cells are intended for implantation in humans.
Primarily for research purposes, there has been some effort to develop biochemically defined media (DM). DM generally includes nutrients, growth factors, hormones, attachment factors, and lipids. The precise composition must be tailored for the specific cell type for which the DM is designed. Successful growth in DM of some cell types, including fibroblasts, keratinocytes, and epithelial cells has been achieved (reviewed by Freshney,1994). However, attachment and proliferation of cells in DM is often not optimal.
One potential application of defined medium is the expansion of chondrocytes released from adult human articular cartilage for treatment of cartilage defects with autologous chondrocyte transplantation (Brittberg et al, New England Journal of Medicine, 331:889-895, 1994). Because this procedure involves the implantation of expanded chondrocytes into a patient, it may be desirable to avoid the use of serum or other undefined components during culture of the chondrocytes. For this application, the DM would need to sustain proliferation of adult human articular chondrocytes seeded at low density until confluent cultures are attained.
Several investigators have reported proliferation of high density non-articular chondrocytes in DM (Kato et al, Exp. Cell Res., 125:167-174, 1980; Madsen et al, Nature, 304:545-547, 1983; Quarto et al, Bone, 17:588, 1995). Others have reported proliferation of rabbit and human articular chondrocytes in DM (Boumedienne et al, Cell Prolif., 28:221-234, 1995; Schwartz, J. Cin. Chem. Clin. Biochem. 24:930-933, 1986). However, in these cases, chondrocytes were tested for growth in DM at high density (.gtoreq.20,000 cells/cm.sup.2). Jennings and Ham (Cell Biology International Reports, 7:149-159, 1983) developed a serum-free medium for proliferation of chondrocytes isolated from costal cartilage of prepubertal humans and seeded at low density. That medium required the use of polylysine-coated plates and included a liposome mixture for which the authors state that there are "inherent limitations in the degree of chemical definition".
Attempts to culture articular chondrocytes at sub-confluent densities in DM have not been successful. Adolphe et al (Exp. Cell Res., 155:527-536,1984) have developed a DM (Ham's F12 supplemented with insulin, transferrin, selenite, fibronectin, bovine serum albumin, brain growth factor, fibroblast growth factor, hydrocortisone, and multiplication stimulating activity--now known as Insulin-like growth factor II) which supports proliferation of rabbit articular chondrocytes. However, they report that serum-containing medium is necessary for the initial attachment of cells to the tissue culture vessel after seeding.
It has been reported that chondrocytes produce and secrete factors that promote their own attachment and proliferation (Shen et al, Endocrinology, 116:920-925, 1985). Examples include basic fibroblast growth factor (Hill et al, Growth Factors, 6:277-294, 1992), insulin-like growth factors (Froger-Gaillard et al, Endocrinology 124:2365-2372, 1989), transforming growth factor-.beta. (Villiger, P.M. et al., J. Immunol., 151:3337-3344, 1993), vitronectin, and possibly some unidentified factors that promote their attachment and proliferation. Because articular cartilage is a non-vascularized tissue, and the chondrocytes embedded in cartilage have limited access to systemic growth factors, autocrine stimulation may play an important role in the maintenance and proliferative capacity of these cells. To our knowledge, autocrine stimulation of chondrocytes has not been utilized for the purpose of enhancing the proliferation of human articular chondrocytes in DM.
During expansion in monolayer in vitro, articular chondrocytes de-differentiate, decreasing synthesis of matrix molecules normally produced by differentiated articular chondrocytes. It has been shown that for cells expanded in serum-containing medium, this process can be reversed by transferring cells to a suspension culture system in the presence of serum (Benya and Shaffer, Cell, 30:215-224, 1982). If cells expanded in DM in monolayer are intended for implantation for healing of cartilage defects (Brittberg et al, 1994), it is important to demonstrate they retain the potential to redifferentiate in suspension culture. A standard procedure for testing for redifferentiation potential is to suspend cells expanded in monolayer into agarose and test for deposition of sulfated glycosaminoglycans by staining with safranin-.largecircle..
A need exists to standardize and control the proliferation and differentiation of adult human articular chondrocytes (HAC) cultured for any medical application, especially for application in humans.